amphotericin-b and thiazolyl-blue

Candida species are known to cause serious fungal infections that produce cutaneous, mucosal, and systemic infections. Nowadays, mortality and morbidity candidiasis in immunocompromised patients have increased. Nanotechnology is a new world-known technology and includes particles ranging from about 1 to 100 nanometers. The purpose of this study was to evaluate the antifungal and cytotoxicity activities of titanium dioxide nanoparticles (TiO2-NPs) and zinc oxide nanoparticles (ZnO-NPs) compared to amphotericin B (AmB) on different Candida spp in in vitro conditions.. In the present study, susceptibility of different Candida species to TiO2-NPs and ZnO-NPs compared to AmB was determined by broth microdilution (BMD) and agar well diffusion methods. Cytotoxicity of TiO2-NPs and ZnO-NPs and amphotericin B was measured by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) assay.. The results indicated that the TiO2-NPs and ZnO-NPs showed antifungal activities against pathogenic Candida spp. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TiO2-NP ranges against Candida spp. were 128-256 µg/mL and 256-512 µg/mL, respectively. The MIC and MFC values of ZnO-NPs were 64-128 µg/mL and 256-512 µg/mL, respectively. However, MICs and MFCs of AmB were 8-16 µg/mL and 16-32 µg/mL, respectively. The MTT assay results showed that the CC50% belonged to ZnO-NPs 706.2 μg/mL, for TiO2-NPs 862.1 μg/mL, and for AmB 70.19 μg/mL, respectively.. Our findings showed that TiO2-NPs and ZnO-NPs had antifungal effects against all Candida species, yet the antifungal properties of TiO2-NPs and ZnO-NPs were significantly less than those of AmB. The CC50% of AmB was significantly lower than ZnO-NPs and TiO2-NPs.

Topics: Amphotericin B; Animals; Antifungal Agents; Candida; Cell Line; Cell Survival; Metal Nanoparticles; Mice; Microbial Sensitivity Tests; Tetrazolium Salts; Thiazoles; Titanium; Zinc Oxide

Orthopaedic fungal infections are commonly treated with systemic amphotericin, which has a narrow therapeutic index and is associated with systemic toxicities. Local delivery of amphotericin has been described yet is poorly understood. As with bacterial infections, fungal infections are associated with biofilm. However, it is unclear whether experience with local delivery of antibacterials can be applied to local antifungal delivery.. We asked whether (1) 100 to 1000 μg amphotericin/mL caused osteoblast cell death; (2) 1 to 10 μg amphotericin/mL caused sublethal toxicity to osteoblasts and fibroblasts; and (3) sublethal amphotericin toxicity could be reversed.. Mouse osteoblasts and fibroblasts were exposed in vitro to amphotericin concentrations of 0, 1, 10, 100, and 1000 μg/mL for 5 hours or 0, 1, 5, and 10 μg/mL for 7 days and then 3 days with no amphotericin. Cell morphology on light microscopy and proliferation assays (alamarBlue(®) and MTT) were used as measures of toxicity.. Amphotericin concentrations of 100 μg/mL and above caused cell death; 5 to 10 μg/mL caused abnormal cell morphology and decreased proliferation. Cells regained normal morphology and resumed cell proliferation within 3 days after removal of amphotericin.. In this in vitro study, amphotericin was cytotoxic to osteoblasts and fibroblasts at concentrations achievable by local delivery.. If local concentrations of 100 to 1000 times the minimum inhibitory concentration are necessary to treat biofilm-associated fungal infections as they are for bacterial infection, cell toxicity at the local depot site should be considered.

Topics: Amphotericin B; Animals; Antifungal Agents; BALB 3T3 Cells; Biofilms; Cell Proliferation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblasts; Mice; Osteoblasts; Tetrazolium Salts; Thiazoles

This study was to investigate whether melatonin (MLT) at pharmacologic concentrations (1 and 100 μM) had potential to influence the expressions of angiogenic (CCL2, CXCL6, IL8) and angiostatic (CXCL10) chemokine genes in two hepatocellular carcinoma (HCC) cell lines with different characteristics (cell line A, HCC24/KMUH, without susceptible to amphotericin B (AmB)-induced oxidative stress; cell line B, HCC38/KMUH, susceptible to AmB-induced oxidative stress). Differential expression of gene was investigated by quantitative reverse transcriptase-polymerase chain reaction. Two genes related to oxidative stress (SOD2, VNN3) were also studied. One and 100 μM MLT up-regulated CCL2, IL8 and CXCL10 genes in cell line A but down-regulated CCL2, CXCL6, IL8 and SOD2 genes in cell line B. CXCL10 gene was up-regulated by 1 and 100 μM MLT in both cell lines. SOD2 gene was down-regulated by 1 and 100 μM MLT only in cell line B. The magnitudes of gene expression fold changes of CCL2 and IL8 genes in cell line A and CCL2, CXCL6, IL8 and SOD2 genes in cell line B were similar between 1 and 100 μM MLT. The magnitudes of gene expression fold change of up-regulated CXCL10 gene in both cell lines were smaller in 100 μM MLT than in 1 μM MLT. In conclusion, the responses of angiogenic chemokine genes to MLT were mainly determined by the characteristics of cancer cells. The concentration of MLT may be the main determinant for the response of angiostatic CXCL10 gene to MLT. Clinical application of MLT in patients with HCC should consider these effects.

Topics: Amidohydrolases; Amphotericin B; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Line, Tumor; Chemokines; Coloring Agents; Down-Regulation; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Liver Neoplasms; Melatonin; Oxidative Stress; Reverse Transcriptase Polymerase Chain Reaction; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Up-Regulation

In light of the need for new antifungals, we compared the in vitro antifungal activity of two peptides derived from human lactoferrin (hLF), i.e., hLF(1-11) and hLF(21-31), two analogs of histatin 5, further referred to as dhvar4 and dhvar5, and two ubiquicidin (UBI)-derived peptides, i.e., UBI 18-35 and UBI 29-41, with that of amphotericin B against Aspergillus fumigatus hyphae using the MTT assay. The results revealed a dose-dependent antifungal activity for all peptides, with dhvar5 being the most potent peptide. In addition, hLF(1-11), dhvar5, and UBI 18-35 were effective against A. fumigatus conidia. Furthermore, hLF(1-11) did not lyze human erythrocytes, whereas dhvar5 (>or=16 microM) and UBI 18-35 (>or=20 microM) were hemolytic. Based on these in vitro results and their effectiveness against infections in mice, we concluded that hLF(1-11) and dhvar5 are promising candidates for the development of new agents against A. fumigatus infections.

Topics: Amphotericin B; Animals; Antifungal Agents; Antimicrobial Cationic Peptides; Aspergillosis; Aspergillus fumigatus; Erythrocytes; Hemolysis; Humans; Hyphae; Mice; Microbial Sensitivity Tests; Microbial Viability; Staining and Labeling; Tetrazolium Salts; Thiazoles

Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimer's and Parkinson's affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimer's disease amyloid (NAC) was found to be an integral part of Alzheimer's dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of alphaSN remains elusive. We have taken the advantage of an alphaSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H(2)O(2), amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that alphaSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of alphaSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, alphaSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the alphaSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimer's and Parkinson's disease.

Topics: alpha-Synuclein; Alzheimer Disease; Amphotericin B; Amyloid; Amyloid beta-Peptides; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Molecular Weight; Neuroblastoma; Peptide Fragments; Ruthenium Red; Tetrazolium Salts; Thiazoles; Time Factors

A total of 27 ethanolic plant extracts from 27 species were screened for leishmanicidal activity in vitro against Leishmania amazonensis. Most of the selected species (19) are traditionally used by the Chayahuitas, an Amazonian Peruvian ethnic group, to treat skin affections and/or leishmaniasis.. A colorimetric method based on the reduction of tetrazolium salt (MTT) was used to measure the viability of Leishmania amazonensis promastigote and amastigote stages.. Only the leaves of two species of the Piperaceae family (Piper hispidum Sw., and Piper strigosum Trel.) showed good leishmanicidal activities (IC(50)<10 microg/ml against amastigotes). Roots of Tabernaemontana sananho Ruiz & Pav. (Apocynaceae), together with bark of Vismia tomentosa Ruiz & Pav. (Clusiaceae), fruits of Solanum straminifolium var straminifolium Jacq. (Solanaceae), and stems of Zamia lindenii Regel ex André (Cycadaceae) showed low activity against amastigote stage (IC(50) around 50 microg/ml). Of those only Tabernaemontana sananho displayed also good activity on promastigotes (IC(50)<10 microg/ml). Results are discussed herein, in relation with the traditional use of the plants and compared with other data from the relevant literature.

Topics: Amphotericin B; Animals; Antiprotozoal Agents; Colorimetry; Drug Evaluation, Preclinical; Ethnobotany; Ethnopharmacology; Humans; Indians, South American; Indicators and Reagents; Leishmania mexicana; Meglumine; Peru; Plant Extracts; Plants, Medicinal; Tetrazolium Salts; Thiazoles

The semi-automated MTT colorimetric assay has previously been applied on Leishmania promastigotes based on the ability of viable parasites to reduce the tetrazolium salt to an insoluble formazan product. As promastigotes are non-adherent, application of the MTT assay in its original form has a major drawback of a high and variable background absorbance due to incomplete removal of phenol red, a component of most media. We have accordingly optimised a modified MTT assay wherein the absorbance linearity was maintained for cells ranging from 1x10(4) to 1x10(7) being 0.04+/-0.003-2.38+/-0.04. In contrast, the original MTT assay had a narrower linearity range of 1x10(6)-1x10(7) cells, absorbances being 0.05+/-0.005-1.54+/-0.005. The modified MTT assay was effectively applied to study growth kinetics and identification of antimonial resistant field isolates. Considering the growing problem of antimonial unresponsiveness in the Indian subcontinent, this modified MTT assay is a useful tool for Leishmania research.

Topics: Amphotericin B; Animals; Antimony Sodium Gluconate; Antiprotozoal Agents; Drug Resistance; Humans; Leishmania donovani; Leishmaniasis, Visceral; Parasitic Sensitivity Tests; Phosphorylcholine; Tetrazolium Salts; Thiazoles

Ravuconazole is a new antifungal triazole with broad-spectrum activity and a long half-life in plasma. We studied the antifungal efficacy, safety, and pharmacokinetics of ravuconazole lysine phosphoester in escalating dosages for the treatment of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Treatment groups consisted of rabbits treated with ravuconazole at 2.5 (RVC2.5), 5 (RVC5), and 10 (RVC10) mg/kg of body weight/day, rabbits treated with amphotericin B (AMB) at 1 mg/kg/day, or untreated controls. There was a dose-dependent reduction of pulmonary residual fungal burden (CFU per gram) in RVC5-, RVC10-, and AMB-treated rabbits in comparison to untreated controls (P < 0.01, P < 0.001, and P < 0.01, respectively). These findings correlated with progressive galactomannan antigenemia in untreated controls and the RVC2.5-treated rabbits, a lower galactomannan index (GMI) in RVC5- and RVC10-treated rabbits, and a similarly low GMI in AMB-treated rabbits (P < 0.01). Rabbits treated with RVC5, RVC10, and AMB also showed a reduction of organism-mediated pulmonary injury, as measured by infarct scores and lung weights, in comparison to untreated controls (P < 0.001). These results were supported by decreased pulmonary infiltrates detected by computed tomography in RVC5- and RVC10-treated rabbits in comparison to untreated controls (P < 0.05). Survival throughout the entire study was achieved in 95% of RVC5-treated rabbits (P < 0.001), 85% of RVC10-treated rabbits (P < 0.001), and 50% of AMB-treated rabbits (P < 0.05) in comparison to none of the untreated controls. Ravuconazole showed linear plasma pharmacokinetics and a large volume of distribution while maintaining concentrations in plasma above the MIC throughout the dosing interval. There was no evidence of hepatotoxicity or nephrotoxicity among ravuconazole-treated animals. Intravenously administered ravuconazole lysine phosphoester showed dose-dependent efficacy and an excellent safety profile for the treatment of invasive pulmonary aspergillosis in persistently neutropenic rabbits.

Topics: Amphotericin B; Animals; Antifungal Agents; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Dose-Response Relationship, Drug; Female; Galactose; Half-Life; Image Processing, Computer-Assisted; Immunosuppressive Agents; Injections, Intravenous; Lung; Mannans; Microbial Sensitivity Tests; Neutropenia; Rabbits; Survival Analysis; Tetrazolium Salts; Thiazoles; Tomography, X-Ray Computed; Triazoles

Heat treatment of deoxycholate-amphotericin B (AmB-DOC) leads to a therapeutically interesting supramolecular rearrangement (h-AmB-DOC); this reformulation improves the therapeutic index of AmB-DOC by reducing amphotericin B (AmB) toxicity in mammalian cell lines from 3- to 10-fold. Its activity in experimentally induced fungal infection in mice remains unchanged compared with AmB-DOC, whereas its activity is 2.5 times higher in Leishmania donovani-infected mice. This work investigates the in vitro mechanism that allows this improvement.. In this study, we analysed the role of serum components on the interaction of h-AmB-DOC with two cultured cell lines: murine peritoneal macrophage cells (J774) and kidney epithelial cells (LLCPK1). The methods used were: spectrophotometry for AmB uptake; MTT assay for cell viability; and lactate dehydrogenase release for membrane damage.. In the presence of 10% fetal calf serum (FCS), the toxicity of AmB-DOC or h-AmB-DOC for both cell lines was null or weak. Interestingly, in J774 cells, the uptake of AmB in the form of h-AmB-DOC was much higher. In LLCPK1 cells, AmB uptake was more limited in both cases but remained higher with h-AmB-DOC. In the absence of FCS, no toxicity for either cell line was observed with h-AmB-DOC.. These findings confirm the importance of serum proteins in AmB biodistribution and suggest that, in vivo, the reduced toxicity and the improved antileishmanial activity of AmB-DOC after moderate heating may be the result of its increased uptake by macrophages.

Topics: Algorithms; Amphotericin B; Animals; Antifungal Agents; Cell Line; Cell Survival; Chemistry, Pharmaceutical; Deoxycholic Acid; Drug Combinations; Hot Temperature; L-Lactate Dehydrogenase; Lipoproteins, LDL; LLC-PK1 Cells; Macrophages; Mice; Swine; Tetrazolium Salts; Thiazoles

Two colorimetric methods that use Alamar Blue or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for assaying the in vitro activities of antifungal agents have been described. We report that both tests performed similarly when the antifungal activity of amphotericin B against Candida albicans was determined. However, only the MTT test generated interpretable data when Aspergillus fumigatus was used.

Topics: Amphotericin B; Antifungal Agents; Aspergillus fumigatus; Candida albicans; Coloring Agents; Dose-Response Relationship, Drug; Microbial Sensitivity Tests; Oxazines; Tetrazolium Salts; Thiazoles; Xanthenes

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30 degrees C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.

Topics: Amphotericin B; Aspergillus fumigatus; Candida albicans; Dose-Response Relationship, Drug; Fluconazole; Formazans; Itraconazole; Microbial Sensitivity Tests; Mitochondria; Tetrazolium Salts; Thiazoles; Vitamin K

The in-vitro influence of cilofungin (LY121019) and amphotericin B on human polymorphonuclear leucocytes (PMNs) was studied by a multifunctional approach. Cilofungin at high concentration (greater than or equal to 20 mg/l) increased adherence to plastic and ingestion of Staphylococcus aureus by PMNs in suspension and Candida albicans by adherent PMNs, and slightly decreased MTT reduction and superoxide generation. Amphotericin B and amphotericin B-deoxycholate decreased adherence to plastic (IC50: 5.1 and 8.2 mg/l respectively) and superoxide generation induced by PMA and opsonized zymosan (IC50 1.1 mg/l for amphotericin B-deoxycholate). Variable results were observed for intra-cellular killing and ingestion. The functional assessment was made with four clinical isolates of yeasts (Can. albicans, Can. tropicalis, Can. Torulopsis) (glabrata, Cryptococcus neoformans). The inoculum was preincubated with the antifungals (PRE) or they were added during (PER) or after ingestion (POST) using PMNs in suspension (PRE and PER), or adhering to plastic (PRE, PER and POST). With the scheme PRE, killing was usually increased with amphotericin B-deoxycholate and cilofungin. With Crypt. neoformans, ingestion was also increased by the antifungals and sodium-deoxycholate, probably by altering the capsule. The results of the scheme POST showed that amphotericin B-deoxycholate, but not cilofungin, increased intracellular killing of Can. albicans, Can. tropicalis and Can. glabrata.

Topics: Amphotericin B; Antifungal Agents; Calcium; Chemotaxis; Deoxycholic Acid; Drug Combinations; Echinocandins; Humans; In Vitro Techniques; Microbial Sensitivity Tests; Neutrophils; Peptides; Peptides, Cyclic; Phagocytosis; Superoxides; Tetrazolium Salts; Thiazoles